High speed, high resolution compositions, methods and kits for capillary electrophoresis

ABSTRACT

The invention provides compositions, methods and kits for high speed, high resolution of analytes by capillary electrophoresis starting with uncoated capillaries. The compositions comprise a sieving component, comprising a non-crosslinked acrylamide polymer, and a surface interaction component, comprising at least one uncharged and non-crosslinked water-soluble silica-adsorbing polymer. Methods for employing the novel compositions in capillary electrophoresis are provided. Kits comprising the novel compositions for use in the novel methods are also provided.

This application is a divisional of U.S. patent application Ser. No.14/921,002, filed Oct. 23, 2015 and now U.S. Pat. No. 9,625,416, whichis a continuation U.S. patent application Ser. No. 13/975,138 filed Aug.23, 2013 and now abandoned; which is a continuation of U.S. patentapplication Ser. No. 13/612,634 filed Sep. 12, 2012 and now abandoned;which is a continuation of U.S. patent application. Ser No. 12/725,425filed Mar. 16, 2010 and now U.S. Pat. No. 8,366,900; which is acontinuation of U.S. patent application Ser. No. 10/794,486 filed Mar.5, 2004 and now U.S. Pat. No. 8,221,607; which is a division of U.S.patent application Ser. No. 09/668,960 filed Sep. 25, 2000 and now U.S.Pat. No. 6,706,162; all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The inventions relate to compositions, capillary electrophoresiselements, and methods for separating analytes by capillaryelectrophoresis. Kits for separating analytes by capillaryelectrophoresis are also provided.

BACKGROUND OF THE INVENTION

Capillary electrophoresis has been applied widely as an analyticaltechnique because of several technical advantages: (i) capillaries havehigh surface-to-volume ratios which permit more efficient heatdissipation which, in turn, permit high electric fields to be used formore rapid separations; (ii) the technique requires minimal samplevolumes; (iii) superior resolution of most analytes is attainable; and(iv) the technique is amenable to automation, see, e.g., Camilleri,editor, Capillary Electrophoresis: Theory and Practice (CRC Press, BocaRaton, 1993); and Grossman et al., editors, Capillary Electrophoresis(Academic Press, San Diego, 1992). Because of these advantages, therehas been great interest in applying capillary electrophoresis to theseparation of biomolecules, particularly nucleic acids. The need forrapid and accurate separation of nucleic acids, particularlydeoxyribonucleic acid (DNA) arises in the analysis of polymerase chainreaction (PCR) products and DNA sequencing, see, e.g., Williams,Methods: A Companion to Methods in Enzymology, 4: 227-232 (1992);Drossman et al., Anal. Chem., 62: 900-903 (1990); Huang et al., Anal.Chem., 64: 2149-2154 (1992); and Swerdlow et al., Nucleic AcidsResearch, 18: 1415-1419 (1990).

Since the charge-to-frictional drag ratio is the same for differentsized polynucleotides in free solution, electrophoretic separation ofpolynucleotides typically involves a sieving medium. The initial sievingmedia of choice were typically crosslinked gels, but in some instancesproblems of stability and manufacturability have led to the examinationof non-gel liquid polymeric sieving media, such as linearpolyacrylamide, hydroxyalkylcellulose, agarose, and cellulose acetate,and the like, e.g., Bode, Anal. Biochem., 83: 204-210 (1977); Bode,Anal. Biochem., 83: 364-371 (1977); Bode, Anal. Biochem., 92: 99-110(1979); Hjerten et al., J. Liquid Chromatography, 12: 2471-2477 (1989);Grossman, U.S. Pat. No. 5,126,021; Zhu et al., U.S. Pat. No. 5,089,111;Tietz et al., Electrophoresis, 13: 614-616 (1992).

Another factor that may complicate separations by capillaryelectrophoresis is the phenomena of electroendoosmosis. This phenomena,sometimes referred to as electroosmosis or electroendoosmotic flow(EOF), is fluid flow in a capillary induced by an electrical field. Thisphenomenon has impeded the application of capillary electrophoresis tosituations where high resolution separations typically are sought, suchas in the analysis of DNA sequencing fragments. The phenomena can arisein capillary electrophoresis when the inner wall of the capillarycontains immobilized charges. Such charges can cause the formation of amobile layer of counter ions which, in turn, moves in the presence of anelectrical field to create a bulk flow of liquid. Unfortunately, themagnitude of the EOF can vary depending on a host of factors, includingvariation in the distribution of charges, selective adsorption ofcomponents of the analyte and/or separation medium, pH of the separationmedium, and the like. Because this variability can reduce one's abilityto resolve closely spaced analyte bands, many attempts have been made todirectly or indirectly control such flow. The attempts have includedcovalent coating or modification of the inner wall of the capillary tosuppress charged groups, use of high viscosity polymers, adjustment ofbuffer pH and/or concentration, use of a gel separation medium forcovalently coating the capillary wall, and the application of anelectric field radial to the axis of the capillary.

Currently, capillary electrophoresis of nucleic acid fragments is oftenperformed using precoated capillaries. Precoated capillary tubestypically are expensive to make, have a limited lifetime, and can besubject to reproducibility problems. These problems are particularlyimportant with large scale capillary electrophoresis using multiplecapillaries run in parallel.

SUMMARY OF THE INVENTION

The present invention provides compositions for separating analytes in asample. For example, single-base resolution of DNA sequencing fragmentsor other polynucleotide fragments. Compositions are provided thatcomprise a sieving component, comprising at least one low viscosity,high molecular weight non-crosslinked acrylamide polymer, andoptionally, a surface interaction component, comprising at least onenon-crosslinked polymer. In a preferred embodiment, the compositions donot comprise a crosslinked polymer gel.

In another aspect, the present invention comprises a capillaryelectrophoresis element. The capillary electrophoresis element comprisesan uncoated capillary into which is inserted a composition forseparating analytes. The composition located within the capillarycomprises a sieving component and a surface interaction component.

In another aspect, methods are provided wherein the compositions of theinvention are employed for separating analytes by capillaryelectrophoresis. In certain embodiments, the methods of the inventionare carried out in parallel using a plurality of uncoated capillaries orcapillary electrophoresis elements containing the novel compositionsdisclosed herein.

In another aspect, the invention provides compositions comprising a lowviscosity, high molecular weight non-crosslinked acrylamide polymersieving component without a surface interaction component for use with,among other things, precoated capillaries. Precoated capillaries arecommercially available, for example, from Bio-Rad Life Sciences (e.g.,Biocap XL capillaries, catalog no. 148-3081). Capillaries may also beprecoated using methods well known in the art. Such procedures aredescribed in, for example, Cobb et al., Anal. Chem. 62:2478 (1990), andGrossman, U.S. Pat. No. 5,347,527.

Kits for separating analytes by capillary electrophoresis are alsoprovided. In certain embodiments, the kits comprise one of thecompositions provided herein. Kits comprising uncoated capillaries foruse with one or more of these compositions or methods are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates single base resolution data collected by capillaryelectrophoresis of labeled polynucleotide molecular weight ladders usingrun temperature of 50° C. and compositions comprising non-crosslinkedacrylamide polymer sieving components with average molecular weights ofapproximately 744,000 daltons (Da) (0.75 M); 1,376,000 Da (1.4 M);2,015,000 Da (2.0 M); 2,517,000 Da (2.5 M); or 6,377,000 Da (6.4 M).FIG. 1A (top) shows a graph of the single base resolution versusfragment size, measured in nucleotide bases. FIG. 1B shows a graph ofthe fragment migration time, in minutes, versus the fragment size,measured in nucleotide bases.

FIG. 2 illustrates single base resolution data collected by capillaryelectrophoresis of labeled polynucleotide ladders using the same fivecompositions described in FIG. 1, but at a run temperature of 60° C.FIG. 2A (top) shows a graph of single base resolution versus fragmentsize, measured in nucleotide bases. FIG. 2B shows a graph of thefragment migration time, in minutes, versus the fragment size, measuredin nucleotide bases.

FIG. 3 illustrates single base resolution data collected by capillaryelectrophoresis of labeled polynucleotide ladders using the same fivecompositions described in FIG. 1, but at a run temperature of 70° C.FIG. 3A (top) shows a graph of the single base resolution versusfragment size, measured in nucleotide bases. FIG. 3B shows a graph ofthe fragment migration time, in minutes, versus the fragment size,measured in nucleotide bases.

FIG. 4 illustrates the single base resolution limit observed usingcompositions comprising the five sieving components described in FIG. 1at run temperatures of 50° C., 60° C., or 70° C. The single baseresolution limits were estimated by visual inspection of the single baseresolution data depicted in FIGS. 1, 2, and 3.

DEFINITIONS

“Acrylamide” and “acrylamide monomer” refers to a structure having theformula H₂C═CR—C(═O)NR₁R₂, where R can be —H or —CH₃, R₁ and R₂ can beindependently —H, —CH₃, —(CH₂)_(x)OH, —CH₂CH(OH)(CH₂)_(y)OR₃,—CH(CH₂OH)CH(OH)CH₃, —CH₂CH₂(OCH₂CH₂)_(p)—OR₃, —CH₂CONH₂,

and R₃ can be independently —H, —CH₃, or —CH₂CH₃. The values for x and yrange from 1 to 3 and the value of p ranges from 1 to 200.

“Average molecular weight” refers to the weight-average molecular weight(M_(w)) of a sample population made up of polymer species having amultiplicity of molecular weights. This quantity is defined by theequation:

$M_{w} = {{\left( {\underset{i = 1}{n_{i}} \times \left( M_{i} \right)^{2}} \right)/\underset{i = 1}{n_{i}}} \times M_{i}}$where n_(i) indicates the number of molecules of species_(i) and M_(i)is the molecular weight of i^(th) species. As used herein, the term“molecular weight” refers to weight average molecular weight, unlessotherwise specified.

The term “capillary” as used herein, refers to a tube or channel orother structure for carrying out electrophoresis that is capable ofsupporting a volume of separation medium, such as a composition forseparating analytes, as disclosed herein. The geometry of a capillarymay vary widely and includes, but is not limited to, tubes withcircular, rectangular or square cross-sections, channels, grooves,plates, and the like, and may be fabricated by a wide range oftechnologies. An important feature of a capillary for use with certainembodiments of the invention is the surface-to-volume ratio of thesurface in contact with the volume of separation medium. High values ofthis ratio typically permit better heat transfer from the separationmedium during electrophoresis. Preferably, in certain embodiments,values in the range of about 0.8 to 0.02 m⁻¹ are employed. Thesecorrespond to the surface-to-volume ratios of tubular capillaries withcircular cross-sections having inside diameters in the range of about 5μm to about 200 μm. The term “uncoated capillary” means that thecapillary is uncoated prior to the introduction of compositions of theinvention, i.e., not covalently coated prior to use. In certainembodiments, capillaries for use with the invention are made of silica,fused silica, quartz, silicate-based glass, such as borosilicate glass,phosphate glass, alumina-containing glass, and the like, or othersilica-like materials. In certain embodiments, capillaries formed inplastic substrates are used. Plastic substrates may comprise, forexample, polyacrylates and polyolefins, such as LUCRYL® (BASF, Germany),TPX™ (Matsui Plastics, Inc., White Plains, N.Y.), TOPAS® (HoechstCelanese Corp., Summit, N.J.), and ZEONOR® (Zeon Chemicals, Louisville,Ky.). Descriptions of plastic substrates for channel capillaries may befound, among other places, in U.S. Pat. No. 5,750,015.

As used herein, the term “composition for separating analytes” comprisesa low viscosity, high molecular weight sieving component and optionally,a surface interaction component. Such compositions are particularlyuseful for separating polynucleotides, or other biomolecules havingdifferent sizes but similar or identical charge-frictional drag ratiosin free solution using capillary electrophoresis. The skilled artisanwill appreciate that a charge-carrying component, or electrolyte istypically included in such compositions. The charge-carrying componentis usually part of a buffer system for maintaining the separation mediumat a constant pH. The compositions for separating analytes contain oneor more non-crosslinked acrylamide polymers.

The term “DNA sequencing fragments” refers to DNA polynucleotidesgenerated for the purpose of obtaining sequence information about aselected DNA target sequence. Such fragments can be generatedenzymatically, e.g., by the Sanger dideoxy method, or chemically, e.g.,by the Maxam and Gilbert method. The fragments may originate in a singlesequencing reaction (e.g., a dideoxy sequencing reaction performed inthe presence of dideoxycytidine tripohophate), or from more than onesequencing reaction (e.g., from four different dideoxy sequencingreactions which include suitably labeled 5′-primers to identify the3′-terminal base of each fragment).

“Polymer” is used in its traditional sense, referring to a largemolecule composed of smaller monomeric or oligomeric subunits covalentlylinked together to form a chain. A “homopolymer” is a polymer made up ofonly one monomeric repeat unit. A “copolymer” refers to a polymer madeup of two or more kinds of monomeric repeat unit. Linear polymers arecomposed of monomeric repeat units linked together in one continuouslength to form polymer molecules. Branched polymers are similar tolinear polymers but have side chains protruding from various branchpoints along the main polymer. Star-shaped polymers are similar tobranched polymers except that multiple side branches radiate from asingle branch site, resulting in a star-shaped or wheel-and-spokeappearance.

Crosslinked polymers contain, for example, polymer molecules that arecovalently linked to each other at points other than at their ends.Crosslinking can occur during the polymerization process in the presenceof crosslinking agents. At some degree of crosslinking, known as the gelpoint, gelation occurs. At the gel point, a visible gel or insolublepolymer forms and the system tends to lose fluidity. This crosslinkedpolymer gel, which corresponds to the formation of a network of polymermolecules that are crosslinked to form a macroscopic molecule, isinsoluble in all solvents, even at elevated temperatures. Discussion ofacrylamide polymers and polymer gels may be found in references known inthe art, for example, Odian, Principles of Polymerization, Third Edition(Wiley Interscience, 1991).

As used herein, the term “non-crosslinked acrylamide polymer” refers topolymer molecules comprising acrylamide monomers, with or withoutbranching, but excluding polymer molecules that are crosslinkedtogether. Thus, a non-crosslinked polymer does not contain polymermolecules that are linked at points other than their end, and does notundergo gelation during polymerization.

The term “polynucleotide” as used herein refers to linear polymers ofnatural or modified nucleoside monomers, including double and singlestranded deoxyribonucleosides, ribonucleosides, -anomeric forms thereof,and the like. Typically, the nucleoside monomers are linked byphosphodiester bonds or analogs thereof to form polynucleotides,however, peptide nucleic acids are also contemplated. In certainembodiments, polynucleotides range in size from a few monomeric units,e.g., 20, to several thousands of monomeric units. Whenever apolynucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′=>3′order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesdeoxythymidine, unless otherwise noted. Analogs of phosphodiesterlinkages include phosphothioate, phosphodithioate, phosphoselenate,phosphodiselenate, phosphoroanilothioate, phosphoranilidate,phosphooramidite, and the like.

The term “single base resolution” (R_(singlebase)) refers to themeasurement of resolution between two peaks arising from twopolynucleotide fragments that differ in size by one nucleotide. Singlebase resolution can be determined mathematically using the formula:

$R_{singlebase} = {2 \times \frac{t_{n} - t_{n + 1}}{W_{n} + W_{n + 1}}}$where t_(n) is the migration time of a polynucleotide fragment that is nnucleotides in length; t_(n+1) is the migration time of a polynucleotidefragment n+1 nucleotides in length; W_(n) is the full width at the baseof the peak from the polynucleotide fragment n nucleotides in length;and W_(n+1) is the full width at the base of the peak from thepolynucleotide fragment n+1 nucleotides in length. “Migration time” isthe time that it takes for a polynucleotide fragment to travel thelength of the capillary or microchannel, i.e., from the injection pointto the detector.

The term “single base resolution limit” refers to the size of apolynucleotide fragment where the single base resolution value dropsbelow 0.58 in a particular system.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference for any purpose to the same extent as if each reference wasspecifically and individually incorporated by reference.

In certain embodiments, the invention provides compositions comprising alow viscosity, high molecular weight non-crosslinked acrylamide polymersieving component. In other embodiments, compositions further comprise asurface interaction component, such as polydimethylacrylamide (pDMA).Furthermore, the compositions of the invention does not include acrosslinked polymer gel. Methods are provided for high speed, highresolution capillary electrophoresis of analytes, particularlypolynucleotide sequences, by using the novel compositions. Kits foremploying these methods are also provided.

One benefit of the compositions of the invention is that non-crosslinkedacrylamide polymers with a molecular weight of between 1,000,000 Da and3,000,000 Da provide unexpected advantages when used in the disclosedmethods and electrophoresis elements compared to known electrophoreticcompositions. Linear acrylamide polymers with molecular weights lessthan about 1,000,000 Da provide poorer resolution than the compositionsof the invention. Linear acrylamide polymers with molecular weightsgreater than about 3,000,000 Da present viscosity problems and are hardto manipulate, e.g., to insert into and remove from capillaries. Thus,the compositions of the invention have a viscosity at 25° C. of lessthan 10,000 centipoise, preferably less than 5,000 centipoise, morepreferably less than 1,000 centipoise, and most preferably less than 600centipoise. Non-crosslinked acrylamide polymers may include, forexample, linear polymers such as polyacrylamide (LPA), branchedpolymers, and star-shaped polymers.

The sieving component may comprise hydrophilic N-substituted acrylamidepolymers (i.e., the substituent is attached to the acrylamide nitrogen)other than polyacrylamide. Exemplary hydrophilic N-substitutedacrylamide polymers include the following homopolymers and theircopolymers:

where R₃ can be —H or —CH₃, R₄ and R₅ can be independently —H, —CH₃,—(CH₂)_(x)OH, —CH₂CH(OH)(CH₂)_(y)OR₆, —CH(CH₂OH)CH(OH)CH₃,—CH₂CH₂(OCH₂CH₂)_(p)OR₆, —CH₂CONH₂,

and R₆ can be independently —H, —CH₃, or —CH₂CH₃. The values for x and yrange from 1 to 3, the value of p ranges from 1 to 200, and q isdirectly proportional to the molecular weight of the polymer and rangesfrom a few hundred to hundreds of thousands. The average molecularweight ranges from 100,000 Da to 25,000,000 Da, preferably from1,000,000 Da to 3,000,000 Da.

One exemplary hydrophilic N-substituted acrylamide copolymer suitablefor use as a sieving component in the disclosed compositions is:

where, R₃, R₄, R₅, and the molecular weight of copolymers, are aspreviously described, and the ratio of m:n ranges from about 100:1 toabout 1:100.

In certain embodiments, the sieving component comprises anon-crosslinked acrylamide polymer having an average molecular weightbetween about 1,000,000 Da and 3,000,000 Da. Non-crosslinked acrylamidepolymers with average molecular weights of 1,000,000 Da or greaterprovide improved resolution. Non-crosslinked acrylamide polymers withaverage molecular weights of 3,000,000 Da or less provide improvedflowability, making such polymers easier to handle and load intouncoated capillaries.

In certain embodiments, the surface interaction component of thecompositions of the invention comprises one or more non-crosslinkedpolymer. Such components may belong to a variety of chemical classes,such as those described in the following references: Molyneux,Water-Soluble Synthetic Polymers: Properties and Behavior, Volumes I andII (CRC Press, Boca Raton, 1982); Davidson, Editor, Handbook ofWater-Soluble Gums and Resins (McGraw-Hill, New York, 1980); Franks,editor, Water: A Comprehensive Treatise (Plenum Press, New York, 1973);and the like.

Exemplary non-crosslinked polymers that may be suitable as a surfaceinteraction component include polyvinylpyrrolidone, N,N-disubstitutedpolyacrylamide, N-monosubstituted polyacrylamides, and the like. Incertain embodiments the surface interaction component comprisespoly(N,N-dimethylacrylamide) (pDMA) in the range of 0.05-0.5%,preferably 0.1-0.4%, and most preferably 0.2%.

Exemplary N-substituents of the N-substituted polyacrylamides include C₁to C₁₂ alkyl; halo-substituted C₁ to C₁₂ alkyl; methoxy-substituted C₁to C₁₂ alkyl; hydroxyl-substituted C₁ to C₁₂ alkyl and the like.Preferably, the halo substituent is fluoro and the hydroxyl-substitutedC₁ to C₁₂ alkyl is monosubstituted. It is understood that the abovemonomer substituents are typically selected so that the resultingpolymer is water soluble. For example, the C₁₂ alkyl-containing monomeris often only present as a small fractional component of a copolymer.More preferably, exemplary substituents are selected from the groupconsisting of C₁ to C₃ alkyl; halo-substituted C₁ to C₃ alkyl;methoxy-substituted C₁ to C₃ alkyl; and hydroxyl-substituted C₁ to C₃alkyl. Such polymers are synthesized by conventional techniques, e.g.,as disclosed in Odian, Principles of Polymerization, Third Edition (JohnWiley, New York, 1991), Glass, editor, Water-Soluble Polymers: Beautyand Performance (Adv. Chem. Ser., #213, American Chemical Society,Washington, D.C., 1986), and Molyneux, Water-Soluble Polymers:Properties and Behavior, Vols. I & II (CRC Press, Boca Raton, Fla.,1982).

A preferred surface interaction component is pDMA. According to certainembodiments, hydrophobic polymers other than pDMA can be used as thesurface interaction component. They include, but are not limited to, thefollowing homopolymers: N-alkyl-substituted acrylamides and theircopolymers,

where R₇ can be —H or —CH₃, R₈ and R₉ can be independently —H, —CH₃,—CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, or —CH₂CONH₂, and z ranges from about2000 to 50,000. The average molecular weight ranges from 200,000 Da to5,000,000 Da, preferably 300,000 Da to 2,500,000 Da. The amide group canalso be cyclic compounds such as

Another example of copolymers that can be used as a surface interactioncomponent include the following structure,

where, R₃, R₄, R₅, and the molecular weight have been describedpreviously, and the ratio of j:k ranges from 1:9 to 9:1.

In certain embodiments, the polymers comprising the surface interactioncomponent of the separation medium may be present at a concentration offrom about 0.001% to about 10% weight:weight (w:w). Preferably, suchpolymers are present at a concentration in the range of about 0.01% toabout 1% w:w.

In certain embodiments, the composition may comprise additionalcomponents such as denaturants. Such denaturants may be useful when itis desirable to prevent the formation of duplexes or secondarystructures, for example, with analytes comprising polynucleotides.Exemplary denaturants include formamide, e.g., 40-90%, urea, e.g., 6-8M, commercially available lactams, such as pyrrolidone, 2-pyrollidinone,and the like. In certain embodiments, denaturants include urea,formamide, or 2-pyrollidinone, alone or in combination. Guidance for theuse of denaturants in electrophoresis can be found in well knownmolecular biology references, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989).

In certain embodiments, the composition has a viscosity of less than10,000 centipoise (cp) at 25° C. In other embodiments the compositionviscosity is less than 5,000 cp at 25° C., less than 1,000 cp at 25° C.,or less than 600 cp at 25° C. All viscosity measurements were made usinga Brookfield Model DV-II viscometer (Brookfield EngineeringLaboratories, Inc., Middleboro, Mass.). For compositions havingviscosities less than 4000 cp, spindle No. 18 was used with the smallsample adapter. The spindle speed was 3 rpm for samples with a viscosityof less than 1000 cp, 1.5 rpm for samples with viscosity of between 1000and 2000 cp, and 0.6 rpm for samples with a viscosity between 2000 and4000 cp. For samples of viscosity over 4000 cp a smaller spindle anddifferent adapter are necessary.

Apparatuses for carrying out capillary electrophoresis are well-known.Many references are available describing basic apparatuses and severalcapillary electrophoresis instruments are commercially available, e.g.,the Applied Biosystems (Foster City, Calif.) model 270A, 310, 3100, or3700 instruments. Exemplary references describing capillaryelectrophoresis apparatus and their operation include Jorgenson,Methods: A Companion to Methods in Enzymology, 4: 179-190 (1992);Colburn et al., Applied Biosystems Research News, issue 1 (winter 1990);Grossman et al. (cited above); and the like.

In certain embodiments, a buffer system is employed to control pH and asa charge-carrying component. Exemplary buffers include: aqueoussolutions of organic acids, such as citric, acetic, or formic acid;zwitterionics, such as TES (N-tris[hydroxymethyl]-2-aminoethanesulfonicacid, BICINE (N,N-bis[2-hydroxyethyl]glycine, ACES(N-[2-Acetamido]-2-aminoethanesulfonic acid), TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid) orglycylglycine; inorganic acids, such as phosphoric; and organic bases,such as Tris (Tris[hydroxymethyl]aminomethane) buffers, e.g., availablefrom Sigma or Calbiochem. Buffer concentration can vary widely, forexample between about 1 mM to 1 M. In certain embodiments, exemplarybuffer solutions for use in the capillary electrophoresis methods of theinvention include: (i) 100 mM TAPS, 7 M urea, pH 8.0; or (ii) TTE (50 mMTris-50 mM TAPS), 7 M urea, pH 8.0.

In certain embodiments, double-stranded polynucleotides, e.g., DNAfragments from PCR or LCR amplifications, enzyme digests, or the like,are separated by standard protocols, or manufacturer's suggestedprotocols where a commercial capillary electrophoresis instrument isemployed, e.g., a model 270-HT, 310, 3100, or 3700 instrument (AppliedBiosystems, Foster City). An exception to such standard or suggestedprotocols is that the compositions and/or capillary electrophoresiselements of the invention are employed. In certain embodiments, a methodfor separating analytes by capillary electrophoresis comprises insertinginto an uncoated capillary, having a first and a second end, acomposition comprising a sieving component and a surface interactioncomponent. A sample of different sized analytes is loaded in thecapillary and an electric field is applied between the first and secondends of the capillary. The different sized analytes in the samplemigrate through the composition within the capillary, separating theanalytes. In other embodiments, a precoated capillary is used. Incertain embodiments, the composition comprises one of the compositionsdisclosed herein.

Certain of the methods of the invention can be employed for DNAsequencing. In certain embodiments, such sequencing involves separationof single stranded polynucleotides prepared by DNA sequencing protocols.Detailed descriptions of DNA sequencing protocols can be found, amongother places, in Automated DNA Sequencing Chemistry Guide (AppliedBiosystems, Part No. 4305080); Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989); Ausbel et al., Current Protocols in Molecular Biology (JohnWiley & Sons, 1993, including supplements through August 2000); or thelike.

An important feature of certain currently available DNA sequencingprotocols is the generation of a “nested series” or “ladder” ofsingle-stranded polynucleotides or DNA sequencing fragments, that may beseparated by size. The chain-termination methods of DNA sequencing maycomprise (1) providing an oligonucleotide primer and a template nucleicacid containing, a target nucleic acid whose sequence is to bedetermined, (2) hybridizing the oligonucleotide primer to the templatenucleic acid, (3) extending the primer with a nucleic acid polymerase,e.g., T7 DNA polymerase, Sequenase™, reverse transcriptase, or the like,in a reaction mixture containing nucleoside triphosphate precursors andat least one chain terminating nucleotide to form a nested series of DNAfragment populations, such that every shorter DNA fragment is asubsequence of every longer DNA fragment and such that each DNA fragmentof the same size terminates with the same chain-terminating nucleotide,(4) separating the DNA fragment populations according to size, and (5)identifying the chain-terminating nucleotide associated with each DNAfragment population. The skilled artisan will appreciate, however, thatmany variations on DNA sequencing methods are available.

Acceptable templates include those discussed in the art, e.g., TechnicalManual for ABI Model 370A DNA Sequencer (Applied Biosystems, FosterCity, Calif.). For example, the target sequence may be inserted into asuitable cloning vector, such as the replicative form of an M13 cloningvector, which is then propagated to amplify the number of copies of thetarget sequence. The single-stranded form of M13 is isolated for use asa template. Also, a template can be provided by polymerase chainreaction (PCR) as taught in the art, e.g., Innis et al., (cited above);Wilson et al., Biotechniques, Vol. 8, pgs. 184-189 (1990); Gyllensten,Biotechniques, Vol. 7, pgs. 700-708 (1989); and the like. Afteramplification, in certain embodiments, the template can be used in thepolymerization reaction(s) either in liquid phase or attached to a solidphase support, e.g., as taught by Stahl et al, Nucleic Acids Research,Vol. 16, pgs. 3025-3038 (1988); Hultman et al., Nucleic Acids Research,Vol. 17, pgs. 4937-4946 (1989); or the like.

Once the nested series DNA fragments are generated, they are separatedby capillary electrophoresis using the compositions, capillaryelectrophoresis elements, or methods of the invention.

The invention, having been described above, may be better understood byreference to examples. The following examples are intended forillustration purposes only, and should not be construed as limiting thescope of the invention in any way.

EXAMPLES Example 1 Preparation of a Non-crosslinked Acrylamide Polymerby Solution Polymerization

A solution containing 94.50 g distilled water (18 M ohm-cm) and 32.02 g(0.129 mol) of a 28.57 wt % acrylamide solution (Bio-Rad, Hercules,Calif.) was prepared in a 500-mL three-necked round bottom flask fittedwith a 2″ Teflon blade for mechanical stirring, a bleeding tube forpurging, and a thermometer. Ultrapure helium (99.99%) was bubbled intothe solution, with constant mechanical stirring, at a rate of 150 mL/minfor 120 minutes, to deoxygenate the solution. To this deoxygenatedsolution, 1.0 mL (13.06 mmol) of 2-propanol (isopropanol) and 4.0 mL(0.35 mmol) of a 1.99 wt % ammonium persulfate (99.99% pure, Aldrich)solution were added with syringes. The flask was immersed in an oil bathat 50±1° C. for 120 minutes with constant mechanical stirring at 150 rpmand helium purging at 150 ml/minute. The reaction was quenched by theaddition of 200 mL of distilled water with stirring and bubbled with airfor 10 minutes. The resulting water-clear solution was placed in aregenerated 50 K molecular weight cutoff (MWCO) Spectra/Por-7 cellulosemembrane and dialyzed against 4.5 gallons of distilled (18 M ohm-cm)water for three days. The water was changed twice daily. The dialyzedsolution was lyophilized and 8.41 g of non-crosslinked acrylamidepolymer was obtained (92% yield).

This polymer was characterized by gel permeation chromatography (GPC)using polyacrylamide primary standards by American Polymer Standards(Mentor, Ohio). Separations were performed at 30° C. with threechromatography columns in series, an Ultrahydrogel (Waters Corp.,Milford, Mass.) 2000 Angstrom column, an Ultrahydrogel 1000 Angstromcolumn, and a guard column, using 0.05M NaNO₃, at a flow rate of 1ml/min. The injection volume was 100 μl and the detector was a KnauerDRI 8×. This non-crosslinked acrylamide polymer was found to have aM_(n) of 589 kilodaltons (kDa), a M_(w) of 2517 kDa (2.5 M in FIGS.1-3), and a polydispersity of 4.23. The M_(w), as determined by batchmode light scattering, was 1936 kDa (Prep. No. 1 in Table 1). Theskilled artisan will appreciate that the observed molecular weight of apolymer may vary depending on the method of characterization. Themolecular weights recited in the appended claims are based on the GPCmethod described above.

The polymer was vacuum dried at 40° C. for at least 4 hours prior touse.

Example 2 Preparation of Another Non-crosslinked Acrylamide Polymer bySolution Polymerization

A second non-crosslinked acrylamide polymer was prepared as described inExample 1, except that 2.0 mL (26.12 mmol) of isopropanol were added tothe deoxygenated solution. The skilled artisan will understand thatisopropanol serves as a chain transfer agent, limiting the molecularweight of the polymer as it is prepared. Thus, by varying the amount ofisopropanol, the molecular weight of the polymer may be altered.

The resulting water-clear solution was dialyzed and lyophilized, as inExample 1. The yield was 9.0 g of non-crosslinked acrylamide polymer(98% yield). The polymer was characterized by gel permeationchromatography and was found to have a M_(n) of 310 kDa, a M_(w) of 1376kDa (1.4 M in FIGS. 1-3), and a polydispersity of 4.40. The M_(w) asdetermined by batch mode light scattering was 975 kDa. (Prep. No. 2 inTable 1).

Following the in the solution polymerization procedure described inExamples 1 and 2, additional non-crosslinked acrylamide polymers withdifferent molecular weights were prepared by varying the concentrationof iso-propanol (see Table 1, Prep. Nos. 1-4).

TABLE 1 Non-crosslinked Acrylamide Polymer Preparations. Mw Mw Prep.Molar ratio Yield (Batch mode (GPC No. [iso-propanol]:[acrylamide] (%)light scattering) method) 1 0.101 92.0 1936 kDa 2517 kDa (2.5M) 2 0.20398.0  975 kDa 1376 kDa (1.4M) 3 0.112 98.0 1325 kDa 2015 kDa (2.0M) 40.406 82.0  697 kDa  744 kDa (0.75M) 5 N/A — 12500 kDa  6377 kDa (6.4M)

Example 3 Preparation of a Non-crosslinked Acrylamide Polymer by InverseEmulsion Polymerization

A fifth non-crosslinked acrylamide polymer was prepared by inverseemulsion polymerization (IEP) as follows. To a 1-L polypropylene beakerwas added 100.05 g of Petrolum Special (bp 180-220° C., Fluka), 100.01 gof a 28.57 wt % acrylamide solution (Bio-Rad), 2.50 g of sorbitanmonooleate (Fluka), and 1.00 mL of a 1.0109 wt % solution of ammoniumpersulfate (99.99%, Aldrich). The mixture was emulsified by stirringwith a 2″ magnetic stir bar for 10 minutes at 800 rpm. The emulsion wasthen transferred into a 1-L three-necked round bottom flask equippedwith a 2″ Teflon stirring blade for mechanical stirring, a bleeding tubefor purging, and a thermometer. The emulsion was purged with ultra purehelium (99.99%) at a rate of 150 mL/min for 120 minutes with constantmechanical stirring at 300 rpm. To the emulsion was added 0.010 mL ofN,N,N,N-tetramethylethylenediamine (ultra pure, Armesco) using amicrosyringe. The flask was lowered into an oil bath at 35±1° C. withconstant mechanical stirring at 300 rpm and helium purging at 150 mL/minfor 19 hours. During polymerization, the temperature of the emulsionnever exceeded 35° C.

After 19 hours, 400 mL of acetone was added and the emulsion was stirredat 300 rpm for 2 hours. The polymer powder was allowed to precipitateand the supernatant layer was decanted. To the precipitated polymer, 300mL of acetone was added, the mechanical stirrer was replaced by a 1.5″egg-shaped magnetic stir bar, and stirred at 800 rpm for 3 hours. Theprecipitated polymer became a very fine powder. This powder was allowedto settle and the organic layer decanted. The polymer powder wastriturated with 300 mL of acetone and stirred at 800 rpm for another 3hours. The polymer was suction filtered and rinsed with a copious amountof acetone. Approximately 5.4 grams of wet polymer powder was added to450 mL of distilled water and the solution was stirred with a 1″magnetic stir bar at 75 rpm for two days. The resulting mixture wasdivided into five 50-mL Falcon tubes and tumbled for two days to yield avery viscous solution. This solution was placed in a regenerated 50KMWCO Spectra/Por-7 cellulose membrane and dialyzed against 4.5 gallonsof distilled water (18 M ohm-cm) for three days. The water was changedtwice daily. The dialyzed solution was lyophilized to give 4.50 g ofnon-crosslinked acrylamide polymer. The M_(w), as determined by batchmode light scattering, was 12500 kDa, and as determined by gelpermeation chromatography, was 6377 kDa (Prep. 5 in Table 1; 6.4 M inFIGS. 1-3).

Example 4 Preparation of Exemplary Sieving Components

Exemplary sieving components for use in the compositions of theinvention were prepared as follows. One hundred milligrams of any of thenon-crosslinked acrylamide polymers shown in Table 1 was added to 2.44grams of water, 0.075 grams of 12.3% pDMA solution, and 0.5 grams of 1 MNa-TAPS/10 mM EDTA buffer, pH 8.0. The mixture was dissolved by rotatingon a rotor wheel overnight. Following this procedure, five differentsieving components, each comprising a different non-crosslinkedacrylamide polymer shown in Table 1, were prepared.

Example 5 Separation of Analytes by Capillary Electrophoresis

Five exemplary compositions for separating analytes were prepared bycombining one of the five sieving components from Example 4 with 0.2%pDMA. These five compositions were analyzed to evaluate their ability toseparate polynucleotide analytes using an ABI 310 capillaryelectrophoresis apparatus (Applied Biosystems, Foster City, Calif.) with47 cm uncoated capillaries (36 cm from the injection end to thedetector). Capillary electrophoresis elements were prepared by pumpingone of the five compositions through uncoated capillaries for 400seconds before each analysis.

Analytes, comprising fluorescently labeled DNA sequencing fragments anda single-stranded DNA sequencing ladder, including 18 DNA fragments ofknown size, labeled with the fluorescent dye TET, were dissolved informamide. This analyte solution was injected into the capillaryelectrophoresis elements at 1.5 kV for 10 seconds. Separations wereperformed at run temperatures of 50° C., 60° C., or 70° C., using anelectric field of 200 V/cm.

The peak width (defined as 4 times the standard deviation of a Gaussianpeak) and migration time of peaks from the DNA sequencing ladder andfragments were measured. These values were used to calculate single baseresolution values. The single base resolution value and migration timefor three or four replicate runs performed at each of the runtemperatures is shown in FIGS. 1, 2, and 3.

The single base resolution limit for the five compositions and the threerun temperatures are shown in FIG. 4. Regardless of run temperature, thesingle base resolution increases as the polymer molecular weightincrease until about 3,000,000 Da (see FIG. 4). Each of the three runtemperature curves plateau, however, when the polymer molecular weightincreases above about 3,000,000 Da. Thus, increasing the molecularweight of the polymer above about 3,000,000 Da does not noticeablyincrease the single base resolution. Increasing the molecular weight ofthe polymer above about 3,000,000 Da does, however, cause a rapidincrease in viscosity of the polymer solution. For example, theviscosity of the 2,500,000 Da polymer used in the Examples wasapproximately 500 centipoise, while the viscosity of the 6,400,000 Dapolymer was greater than 50,000 centipoise. Thus, sieving compositionswith molecular weights of 1,000,000 to 3,000,000 Da provide efficientsingle base resolution while retaining sufficiently low viscosity forcapillary loading.

Although the invention has been described with reference to variousapplications, methods, and compositions, it will be appreciated thatvarious changes and modifications may be made without departing from theinvention. The foregoing examples are provided to better illustrate theinvention and are not intended to limit the scope of the invention.

What is claimed is:
 1. A method for separating analytes by capillaryelectrophoresis comprising: placing the analytes in a compositioncomprising: a sieving component comprising a non-crosslinked acrylamidepolymer having a weight average molecular weight between about 3,000,000Daltons (Da) Da and less than or equal to about 6,400,000 Da; and asurface interaction component comprising one or more non-crosslinkedpolymers selected from the group consisting of poly(meth) acrylamide,N,N-disubstituted polyacrylamide and N-substituted polyacrylamide,wherein the N-substituents are selected from the group consisting of C₁to C₃ alkyl, halo-substituted C₁ to C₃ alkyl, methoxy-substituted C₁ toC₃ alkyl, and hydroxyl-substituted C₁ to C₃ alkyl; wherein the sievingcomponent and the surface interaction component are the same ordifferent; and wherein the composition does not include a cross-linkedpolymer gel; and separating the analytes by capillary electrophoresis.2. The method of claim 1, which is carried out in parallel with aplurality of uncoated capillaries.
 3. The method of claim 1, wherein thecomposition further comprises at least one denaturant.
 4. The method ofclaim 3, wherein the at least one denaturant is selected from the groupconsisting of at least one of formamide, urea and 2-pyrollidinone. 5.The method of claim 4, wherein the at least one denaturant comprisesurea.
 6. A method for separating analytes by capillary electrophoresiscomprising: separating the analytes by capillary electrophoresis with acapillary electrophoresis element comprising: an uncoated capillary; acomposition for separating analytes located within the uncoatedcapillary, the composition comprising: a sieving component comprising auncrosslinked acrylamide polymer having a weight average molecularweight between about 3,000,000 Daltons (Da) and less than or equal toabout 6,400,000 Da; and a surface interaction component; wherein thesurface interaction component comprises a solution of one or morenon-crosslinked polymers; and wherein the capillary electrophoresiselement does not include a crosslinked polymeric gel.
 7. The method ofclaim 6, which is carried out in parallel with a plurality of uncoatedcapillaries.
 8. The method of claim 7, wherein the uncoated capillariescomprise silica, fused silica, quartz, silicate-based glass, phosphateglass, or alumina-containing glass.
 9. The method of claim 7, whereinthe uncoated capillaries comprise a plastic substrate.
 10. A method forseparating analytes by capillary electrophoresis comprising: insertinginto an uncoated capillary having a first end and a second end acomposition comprising a sieving component comprising an uncrosslinkedacrylamide polymer having a weight average molecular weight betweenabout 3,000,000 Da and less than or equal to about 6,400,000 Da and asurface interaction component comprising one or more non-crosslinkedpolymers selected from the group consisting of poly(meth) acrylamide,N,N-disubstituted polyacrylamide and N-substitute polyacrylamide,wherein the N-substituents are selected from the group consisting of C₁to C₃ alkyl, halo-substituted C₁ to C₃ alkyl, methoxy-substituted C₁ toC₃ alkyl, and hydroxyl-substituted C₁ to C₃ alkyl; wherein the sievingcomponent and the surface interaction component are the same ordifferent; wherein the compositions does not include a crosslinkedpolymer gel; loading a sample of different sized analytes into thecapillary; and applying an electrical field between the first and secondends of the capillary so that the different sized analytes in the samplemigrate through the capillary, thereby separating the analytes.
 11. Themethod of claim 10, wherein the composition further comprises at leastone denaturant.
 12. The method of claim 11, wherein the at least onedenaturant comprises urea.
 13. The method of claim 10, wherein thesurface interaction non-cross-linked polymer ispoly(N,N-dimethylacrylamide).
 14. The method of claim 10, which iscarried out in parallel with a plurality of uncoated capillaries. 15.The method of claim 14, wherein the uncoated capillaries comprisesilica, fused silica, quartz, silicate-based glass, phosphate glass, oralumina-containing glass.
 16. The method of claim 14, wherein theuncoated capillaries comprise a plastic substrate.